Post-mitotic neurons can enter into the cell cycle after stroke but die instead of proliferating. Our data showed up- regulation of cyclin-dependent kinase 4 (Cdk4) after oxygen-glucose deprivation (OGD)/reoxygenation in near-pure primary cortical neuronal cultures, evidence of cell cycle entry. After cerebral ischemia, expressions of cell cycle proteins are altered. Inhibiting the cell cycle after stroke will provide benefit by attenuating neuronal death and proliferation of microglia/macrophages. Cell cycle regulation by lipid second messengers after stroke: Sphingomyelin (SM) synthase (SMS) transfers phosphocholine from phosphatidylcholine (PC) to ceramide to form SM and 1,2- diacylglycerol (DAG). Ceramide and DAG are key regulators of the cell cycle and altering their formation affects both neuronal and non-neuronal cell fate after stroke. Tricyclodecan-9-yl-xanthogenate (D609) inhibits SMS leading to ceramide accumulation. Ceramide can induce cell cycle arrest by (a) activating protein phosphatases 1 and 2A (PP1 and PP2A), (b) dephosphorylation of retinoblastoma (Rb) and Cdk2 and (c) up-regulation of Cdk inhibitors p21 and p27. Hypothesis: D609 may block the cell cycle, attenuating neuronal death and non-neuronal cell proliferation by increasing ceramide levels after stroke. The effect of D609 on ceramide de novo synthesis pathway will also be examined. In support of our hypothesis, D609 (a) significantly reduced cerebral infarction at reperfusion days 1 and 3, (b) up-regulated p21 and p27 through ceramide accumulation, and (c) attenuated Rb phosphorylation after transient middle cerebral artery occlusion (tMCAO) in rat. Our studies strongly support SMS inhibition by D609 leading to cell cycle arrest. To understand D609 mechanism, the following aims will test the hypothesis: Aim 1: Does D609 inhibit SMS, blocking the cell cycle and providing protection after OGD/reoxygenation in near-pure primary cortical neuronal cultures? Our studies showed that D609 up-regulated p27 in primary neuronal cultures after OGD/reoxygenation, suggesting increased ceramide due to SMS inhibition. Aim 2: How does SMS regulate cell cycle proteins and proliferation of RAW 264.7 macrophages? In vitro silencing of SMS (both in neuronal and macrophage cultures, Aims 1-2) will confirm the actions of D609 mediated through inhibition of SMS. Aim 3: How does D609 regulate SM metabolism, expression of cell cycle proteins, and microglia/macrophage proliferation in rat tMCAO? Our data suggest that D609 neuroprotection is due to increased ceramide levels, up-regulation of p21 and cell cycle arrest after tMCAO. Microglia/macrophages are the primary source of TNF-1 and IL-1 that are rapidly up-regulated after stroke and contribute to brain injury. We anticipate that D609 will reduce proliferation of microglia/macrophages as well as TNF-1 and IL-1ss expression after tMCAO, providing benefit. In vivo SMS silencing and SMS2 conditional (neuron-specific) knockout using cre/loxP system are proposed as alternatives. Translational potential: tPA has limited use in stroke patients. Although it is premature to predict, lipid metabolites that affect the cell cycle system in stroke have not been extensively studied and have not undergone stroke clinical trials. This proposal explores the therapeutic potential of D609 and how it affects lipid second messenger ceramide that regulates the cell cycle both in vitro and in vivo stroke models. PUBLIC HEALTH RELEVANCE: Stroke is a worldwide health care concern and a leading cause of disability. In the USA;healthcare costs are >$63 billion/year. Currently FDA approved tPA has a very limited use in stroke patients. The disappointing NXY-059 stroke clinical trials emphasized the need for new treatments. This research seeks to modulate a lipid metabolite by pharmacologically altering an enzyme system(s) that may offer benefit and may provide clues to develop lead molecules. The long-range thrust of this research is to develop strategies to minimize disabilities due to stroke.